The chemical composition of surfaces plays a pivotal role in dictating the overall efficacy of many devices. Applications in which surface chemistry exerts a major influence in device performance include the biocompatibility of materials, biosensors, heterogeneous catalysis, and the permselectivity of membranes, to mention but a few of the many such examples. In recognition of the important role exerted by surfaces, surface modification research has represented an exceedingly active area for many years. A wide range of techniques, involving both physically and chemically oriented approaches, has been developed in efforts to provide specific improvements of surfaces. Examples of such work include various vapor deposition methods, plasma processes, sputtering techniques, chemical etching processes, ion implantation, etc. In these processes, the surface treatments are designed to improve device performance and/or reduce costs involved in preparation of substrate surfaces. An enormous patent literature exists describing the various surface modification techniques which have been developed and the many applications which have been identified.
Despite the extensive work in this area, a significant need remains for improved methods to control surface modification processes at the molecular level. Unfortunately, the majority of currently employed surface modification techniques provide unsatisfactory controllability of the film chemistry during the deposition process. Additionally, current surface modification techniques, as employed on industrial scale operations, are restricted to planar, 2-dimensional surface layers.
In recognition of the need for improved molecular level film chemistry control, and particularly the requirement for three-dimensionally molecularly designed surfaces, additional approaches to surface modification have been described in recent literature. For example, the use of self-assembling monolayers (SAM's) have been extensively developed during recent years to provide layered structures having three-dimensional molecular properties. A second developing approach to surface modifications, and one of direct relevance with respect to the present invention, is use of an initial surface treatment to introduce reactive functional groups which are then subjected to subsequent chemical derivatization processes. These multi-step procedures, ultimately resulting in coupling of specific molecules to the surface, can provide what may be described as three-dimensional molecularly tailored surface.
Although these new surface modification procedures have produced some interesting results, the overall processes described to date are exceedingly time-consuming, inefficient and require the use of undesirable hazardous solvents. Descriptions of recent literature reports of these surface modification processes clearly reveal the complex procedures involved in attempting to couple specific molecules to surfaces. For example, recent work has employed a plasma deposition method for introduction of surface hydroxyl (--OH) groups, which are then reacted in a second step to provide covalently bonded molecules tethered to the surface (Ranieri et al.). In this process, the plasma-introduced surface --OH groups are first reacted with di-imidazole, with this reaction being carried out in dry tetrahydrofuran at room temperature for 30 minutes. This was then followed by an aqueous solution reaction at 4.degree. C. for 72 hours at a pH of 8.4 to covalently attach low molecular weight peptides to the surface. This process requires both the use of an nonaqueous solvent and an extraordinarily long (i.e., 72-hour) coupling reaction to couple the desired molecules to the surface (Ranieri et al.).
Alternately, a chemical process can be employed to introduce the surface hydroxyl groups which are then subsequently derivatized. For example, a complex procedure has been employed to provide a so-called glycol phase surface which contains reactive --OH groups (Massia et al.). In this procedure, glass coverslips are initially soaked in 0.5 M NaOH for 2 hours, then rinsed in deionized water and then immersed in pH 5.5 aqueous solution of (3-glycidoxypropyl)trimethoxy-silane. This reaction was maintained for 2 hours after which time the pH was adjusted to 3.0 followed by heating again for 1 hour to convert the oxirane moieties on the derivatized glass to glycol groups. This was followed by reaction of the surface hydroxyls with tresyl chloride using an acetone solvent followed by rinsing with 1 mM HCI and immersion in 0.2 M NaHCO.sub.3 buffer at pH 9. Coupling of the desired peptides to the glass surface was then carried out via a 20-hour incubation reaction to the sulfonyl-containing surfaces.
Still other workers have employed a somewhat different but equally complex route to obtain the glycophase glass surface (Clemence et al.). In this work, they employ a sequence in which the glass coverslips are incubated for 5 minutes in a boiling solution of NH.sub.3 /H.sub.2 O.sub.2 /H.sub.2 O. After rinsing with distilled water, the glass disks were rinsed in acetone after which they were reacted with a solution containing CPTMS and TEA in dry toluene. This was followed by successive washes in chloroform, acetone and methanol and then dried in vacuum. This was followed by a HCl rinse, then incubation for 60 min. at 90.degree. C. in 1 mM HCl followed by rinsing with doubly distilled water to obtain the glycophase glass. These workers then employed a photochemical method to attach peptides covalently to the hydroxylated surfaces. This technique requires the initial coupling of a photochemical label to the peptides. The photochemical labels employed were N-{m-[3-(trifluoromethyl)-diazirin-3-yl]phenyl}-4-maleimidobutyramide or 4-maleimidobenzophenone. With either label a complicated reaction procedure is required to attach them to the peptide with the synthesis requiring the use of HPLC to separate the desired complex from the reaction mixtures. These photolabeled molecules are then subsequently attached to hydroxylated surfaces using a photochemical technique.
Alternately, biomolecule surface immobilization has been reported using bifunctional photochemical sensitive molecules to achieve this goal (Sigrist et al.). In this approach, a heterobifunctional crosslinker is employed to link the biomolecules to a surface. For example, 3-(trifluoromethyl)-3-(m-isothiocyanophenyl) diazirine is initially coupled to a surface containing amine groups via coupling through the isothiocyano group. Subsequent photolysis of the surface attached diazirine generates a carbene radical which can react with biomolecules, if the biomolecules are within molecular vicinity at the time of carbene generation. As noted by these authors: "If target molecules are not present during the carbene lifetime, the intermediate will react with every molecular species present including water." This of course leads to relatively low surface immobilization of the protein molecules as the process selectivity is relatively low. Thus this recent biomolecule surface immobilization process requires synthesis of a complex heterobifunctional photochemical sensitive intermediate linker molecule, attachment of this linker to a functionalized surface and, finally, photochemical attachment of this linker to the biomolecules. Over all the process is complex and it results in relatively low yields and low selectivity of biomolecule attachment to surfaces.
The above results have been cited to document the relatively complex reaction procedures currently being employed to covalently attach molecules to surfaces. The research cited above is from leading researchers and includes literature citations as recent as 1995. Thus it seems accurate to conclude that the complex techniques employed represent "state-of-the-art" in surface modifications in which molecules are attached to activated surfaces.
The process of the present invention is to be contrasted with numerous earlier applications of plasma surface modifications to enhance the interaction of solid substrates with other molecules and materials. Earlier plasma depositions were employed to improve the adsorption and/or adhesion of various molecules to the modified surfaces. For example, U.S. Pat. Nos.5,055,316 (Hoffman et al.) and 5,258,127 (Gsell et al.) both employed plasma surface modification to enhance adsorption of various biomolecules. In a similar vein, U.S. Pat. No. 5,178,962 (Miyamoto et al.) utilized a plasma discharge process to change the chemistry of a macromolecular synthetic resin film by exposure to excited plasma species to generate surface active groups, which are then coupled to metal atoms to form a metallic outer layer. This latter work involved non-polymerizable gases and the metal films were deposited on the plasma activated surfaces by high energy vapor deposition processes.
One way that the present invention differs from the above noted techniques in that the initial plasma surface modification step is directly followed by a chemical derivatization process in which desired molecules are covalently bound to the surfaces via simple chemical reaction.